Calcium release-activated Calcium (CRAC) current: Impact on human B-cell function and its physiological role in human mesothelial cells

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Calcium (Ca2+) is an ubiquitous signalling messenger and plays an essential role in every aspect of a cell s life and death. Cells can increase their intracellular Ca2+ concentration either by releasing Ca2+ from intracellular stores or by opening of Ca2+ conducting channels in the plasma membrane (PM), such as the Calcium release-activated calcium channel (CRAC) composed of the channel-forming protein Orai (after keeper of heaven's gate in Greek mythology) and the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule (STIM). Upon store depletion, STIM binds and activates plasma membrane localized Orai channels, providing a Ca2+ influx pathway that is essential for proper cell function in almost all eukaryotic cells. Careful titration of the protein concentration of STIM and Orai thus determines the amount of Ca2+ influx and its subsequent downstream effects e.g. nuclear factor of activated T cells (NFAT) signalling cascades of gene transcription, proapoptotic pathways such as EK signalling or survival pathways such as B/Akt. The channel forming protein Orai exists as the three homologs Orai1, 2 and 3. The calcium sensing protein STIM exists as the two homologs STIM1 and 2. It is already shown that store operated calcium entry (SOCE) plays a crucial role in immune cell function and that dysregulation in this process leads to the development of immunodeficiency, autoimmunity and cancer.While the physiological role of Orai1 was intensively researched within the last years, the impact of Orai2 or Orai3 function in primary cells remains still elusive. Thus, I propose to investigate the physiological role of Orai2 in primary human B-cells and B-cell lines (Daudi, Raji). Human B-cells exhibit a perfect model system for the delineation of the native Orai2 function, expressing remarkable amounts of Orai2 and easy to isolate and to handle. I will characterize the Orai mediated Ca2+ influx with electrophysiological, Fura2-based imaging and silencing approaches and investigate its physiological role regarding the antibody production or the interleukine secretion e.g. IL-10. This part of the project will gain our limited knowledge about Orai2 in general and about CRAC/SOCE in human B-cells (SA 1). Ca2+ signalling and the CRAC current play also a crucial role in my second research project. The TNFalpha and NF-kappaB signalling pathways are both Ca2+ dependent processes. Haining Yang et al. were able to identify the importance of these signalling pathways in the genesis of asbestos-induced mesothelioma, a special form of cancer, which causes ~2.000-3.000 deaths per year in the U.S.. The underlying Ca2+ mobilizing processes are still elusive and in general it is almost nothing known about Ca2+ signalling in human mesothelial cells (HM). So I suggest to characterize SOCE and CRAC in primary human mesothelial cells and to elucidate the contribution of this specific Ca2+ entry pathway to the development of asbestos-induced oncogenesis (SA 2).